title: Purification of yeast alcohol dehydrogenase by using immobilized metal affinity cryogels.
abstract: In this study, poly(2-hydroxyethyl methacrylate-glycidylmethacrylate) [poly(HEMA-GMA)] cryogels were prepared by radical cryocopolymerization of HEMA with GMA as a functional comonomer and N,N'-methylene-bisacrylamide (MBAAm) as a crosslinker. Iminodiacetic acid (IDA) functional groups were attached via ring opening of the epoxy group on the poly(HEMA-GMA) cryogels and then Zn(II) ions were chelated with these structures. Characterization of cryogels was performed by FTIR, SEM, EDX and swelling studies. These cryogels have interconnected pores of 30-50 μm size. The equilibrium swelling degree of Zn(II) chelated poly(HEMA-GMA)-IDA cryogels was approximately 600%. Zn(II) chelated poly(HEMA-GMA)-IDA cryogels were used in the adsorption of alcohol dehydrogenase from aqueous solutions and adsorption was performed in continuous system. The effects of pH, alcohol dehydrogenase concentration, temperature, and flow rate on adsorption were investigated. The maximum amount of alcohol dehydrogenase adsorption was determined to be 9.94 mg/g cryogel at 1.0mg/mL alcohol dehydrogenase concentration and in acetate buffer at pH5.0 with a flow rate of 0.5 mL/min. Desorption of adsorbed alcohol dehydrogenase was carried out by using 1.0M NaCI at pH8.0 phosphate buffer and desorption yield was found to be 93.5%. Additionally, these cryogels were used for purification of alcohol dehydrogenase from yeast with a single-step. The purity of desorbed alcohol dehydrogenase was shown by silver-stained SDS-PAGE. This purification process can successfully be used for the purification of alcohol dehydrogenase from unclarified yeast homogenates and this work is the first report about the usage of the cryogels for purification of alcohol dehydrogenase.
authors.affiliation: Chemistry Department, Adnan Menderes University, Aydın, Turkey.
authors.forename: Begüm
authors.initials: B
authors.lastname: Akduman
authors.forename: Murat
authors.initials: M
authors.lastname: Uygun
authors.forename: Deniz Aktaş
authors.initials: DA
authors.lastname: Uygun
authors.forename: Sinan
authors.initials: S
authors.lastname: Akgöl
authors.forename: Adil
authors.initials: A
authors.lastname: Denizli
mesh_terms: D000327:Adsorption; D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D005782:Gels / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007098:Imino Acids / Q000737:chemistry; D011073:Polyamines / Q000737:chemistry; D011102:Polyhydroxyethyl Methacrylate / Q000031:analogs & derivatives / Q000737:chemistry; D011109:Polymethacrylic Acids / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013696:Temperature; D015032:Zinc / Q000737:chemistry*
chemical_list: D005782:Gels; D007098:Imino Acids; D011073:Polyamines; D011109:Polymethacrylic Acids; C056971:poly(2-hydroxyethyl methacrylate)-polyamine graft copolymer; C042535:polyglycidyl methacrylate; D011102:Polyhydroxyethyl Methacrylate; D000426:Alcohol Dehydrogenase; D015032:Zinc; C008109:iminodiacetic acid
keywords: Alcohol dehydrogenase; Cryogel; IMAC; Iminodiacetic acid; Yeast